Project Summary The mouse mammary gland provides a robust mammalian system for investigating how epithelial behavior impacts tumorigenesis. The mammary epithelium is composed of an inner layer of milk-producing luminal cells and an outer layer of contractile myoepithelial cells. Several mammalian epithelial tissues rely on oriented cell divisions to maintain proper structure and function, however it remains unclear whether this mechanism plays the same important role in the mammary gland. I hypothesize that misoriented cell divisions in the luminal layer drive cell escape from the epithelium and promote breast cancer initiation. To test this hypothesis, I will first force luminal cells to divide out of the epithelial plane by manipulating spindle orientation mechanisms both in vivo and in 3D organoid culture. I will determine whether this intervention causes luminal filling through apically-oriented cell escape, invasive behaviors through basally-oriented escape, hyperproliferation and/or apoptosis. In my second approach, I will employ human and mouse breast cancer models to both analyze spindle orientation and determine whether misoriented divisions are driven by oncogenic activation. These studies could ultimately reveal important therapeutic targets for preventing, detecting and treating invasive breast cancers.